Quantitative Peptide or Protein Assay

ABSTRACT

Peptide and/or protein quantitation methods, kits, and compositions, particularly useful for mass spectrometry, are provided herein based on a bathocuproine-based composition complex such as bathocuproinedisulfonic acid disodium salt hydrate complex. The methods are one-step rapid absorbance methods using small sample volumes. They produce a robust signal with high signal to background ratio and accurately quantitate even complex peptide mixtures with low variability and high sensitivity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/299,667, filed Mar. 12, 2019, which is a divisional of U.S. patentapplication Ser. No. 15/674,831, filed Aug. 11, 2017, now abandoned,which is a divisional of Ser. No. 14/734,678, filed Jun. 9, 2015, nowabandoned, which claims priority to U.S. Provisional Patent ApplicationNo. 62/010,594 filed Jun. 11, 2014, the entirety of which isincorporated by reference herein.

FIELD

Peptide and/or protein assay methods, compositions, and kits, useful fordetermining the concentration of small volume samples before analysis bymass spectrophotometry (MS) and other types of analyses.

BACKGROUND

Mass spectrometry (MS) is a sensitive method for the simultaneousidentification and relative quantitation of thousands of proteinsbetween multiple samples. On-going advancements in MS instrumentationcontinue to push the ability of the scientific community to characterizethe protein dynamics in complex biological systems to a great depth.Despite MS capabilities, a majority of MS samples are analyzed withoutsignificant pre-injection characterization or normalization, becausecurrent methods to monitor and measure proteins, such as UV absorptionor bichinconic acid (BCA) assays, work poorly with peptides, consume toomuch valuable sample, and lack the required sensitivity. This lack ofsample characterization and normalization leads to difficulties withstandardization and reproducibility of MS experiments and significantunder-productive instrument time.

Commercially available colorimetric protein and peptide solutionquantitation methods include biuret (Gornall et al. J. Biol. Chem. 177(1949) 751), Lowry (Lowry et al. J. Biol. Chem. 193 (1951) 265),bicinchoninic acid (BCA) (Smith et al. Anal. Biochem. 150 (1985) 76),Coomassie Blue G-250 dye-binding (Bradford, Anal. Biochem. 72 (1976)248), and colloidal gold (Stoscheck, Anal. Biochem. 160 (1987) 301).

The biuret method is based on a protein forming a complex with cupricions. Peptide nitrogen binds to copper (II) ion under alkalineconditions, producing a purple color. The absorption maximum of theproduct is 550 nm. The sensitivity is 1 mg protein/ml to 6 mgprotein/ml. The biuret method is a relatively insensitive proteindetermination method compared to other commercial methods ofcolorimetric protein determination.

Another method combines the biuret reaction and thecopper(1)-bathocuproine chelate reaction (Determination of Proteins by aReverse Biuret Method Combined with the Copper-Bathocuproine ChelateReaction. Clinica Chimica Acta., 216 (1993) 103-111). In this method, asample protein forms a Cu²⁺-protein chelate complex (biuret reaction)during the first step. Excess Cu²⁺ is reduced to Cu⁺ by ascorbic acid,allowing Cu⁺ to form a Cu⁺-bathocuproine chelate complex during thesecond step. The amount of Cu⁺-bathocuproine chelate complex formed isinversely proportional to the protein concentration. This is a negativeor indirect assay using a bathocuproine chelator to determine proteinconcentration.

The Lowry method is a modified biuret reaction. It occurs in two steps:first, peptide bonds react with copper(II) ions under alkalineconditions, then Folin-Ciocalteau phosphomolybdic-phosphotungstic acidreduces to heteropolymolybdenum blue by copper-catalyzed oxidation ofaromatic amino acids. The absorption maximum of the product is 750 nm.The Lowry method is more sensitive than the biuret method, with a linearsensitivity of 0.1 mg protein/ml to 1.5 mg protein/ml for bovine serumalbumin (BSA). Certain amino acids, detergents, lipids, sugars, andnucleic acids interfere with the reaction. The reaction is pH dependentand pH should be maintained between pH 10 and pH 10.5.

The BCA method is related to the Lowry method in that peptide bonds inproteins first reduce cupric ion (Cu²⁺) to produce atetradentate-cuprous ion (Cu¹⁺) complex in an alkaline medium. Thecuprous ion complex then reacts with BCA (2 molecules BCA per Cu¹⁺) toform an intense purple color that can be measured at 562 nm. TheBCA-Copper reaction is shown below:

Because BCA is stable in alkaline medium, the BCA method can be carriedout in one step, compared to two steps needed in the Lowry method. TheBCA method better tolerates potential inhibitory or interferingcompounds in the sample compared to the Lowry method. For example, up to5% of each of sodium dodecyl sulfate (SDS), Triton X-100, and Tween-20can be present and not interfere with the BCA method, compared to only1% SDS, 0.03% Triton X-100, and 0.062% Tween-20 that can be present andnot interfere with the Lowry method. The BCA method also has increasedsensitivity and an expanded linear working range compared to the Lowrymethod.

A MICRO BCA™ Protein Assay Kit (Thermo Fisher Scientific) permitsquantitation of dilute sample solutions (0.5 μg/ml to 20 μg/ml) by usinglarger sample volumes to obtain higher sensitivity. Despite theincreased sensitivity, sample volume requirements limit or prevent itsuse for quantitation of many peptide samples.

A modified BCA assay to quantitate peptides (Kapoor et al. AnalyticalBiochemistry, 393 (2009) 138-140) acknowledges difficulties measuringpeptide concentrations because of high interpeptide variation largelybecause of peptide hydrophobicity. The modified BCA method estimatespeptide concentration by denaturing peptides by treatment at 95° C. forfive minutes in the presence of SDS prior to incubation with the BCAworking reagent. However, data below 500 μg/ml is very close to noiselevel and thus is not reliable.

U.S. Pat. No. 4,839,295 discloses using bicinchoninic acid as a chelatorto detect proteins, measuring absorbance at 562 nm.

The colloidal gold method is the most sensitive among the colorimetricprotein determination methods. Its sensitivity is about 2 μg/ml to 20μg/ml protein. However, there is significant protein-to-proteinvariation. Protein binding to colloidal gold causes a shift in colloidalgold absorbance that is proportional to the amount of protein insolution. Most common reagents other than thiols and sodium dodecylsulfate (SDS) are compatible with the colloidal gold method.

The Coomassie Blue G-250 dye-binding method is based on the immediateabsorbance shift from 470 nm to 595 nm that occurs when Coomassie BlueG-250 binds protein in an acidic medium. Color development is rapid andthe assay can be performed in ten minutes. The Coomassie Blue G-250dye-binding method is comparatively free from interference by commonreagents except detergents. There is moderate protein-to-proteinvariation and the method does not work well with peptides.

A total protein assay (Sozgen et al., Talanta, 68 (2006) 1601-1609Spectrophotometric total protein assay with copper (II) neocuproinereagent in alkaline medium) uses copper(II)-neocuproine (Nc) reagent inalkaline medium with a hydroxide-carbonate-tartarate solution, withneocuproine as chelator. After 30 min incubation at 40° C., absorbanceof the reduction product, Cu(I)-Nc complex, is read at 450 nm against areagent blank. This assay has limited sensitivity because of the limitedsolubility of neocuproine in alkaline aqueous solution.

U.S. Pat. No. 5,693,291 discloses a quantitative protein method. Themethod is an indirect two-step method. It uses two reagents: reagent A(tartrate solution and copper sulfate) and reagent B (reducing agent,e.g., ascorbic acid, and bathocuproine chelator). Reagent A contains 0.7to 2 mmol/l Cu²⁺ ions and 2 to 4 mmol/l tartrate in alkaline solution.Reagent B contains 1 to 1.5 mmol/l ascorbic acid and 0.5 to 0.8 mmol/lbathocuproine. The proportion of reagent A to reagent B is 1:8 to 1:12,i.e., 1 part reagent A to 8-12 parts reagent B. The combined volume ofreagent A and reagent B is between 750 μl and 3000 μl, which isrelatively large. Step one of the method mixes 100 μl Reagent A to 50 μlsample, followed by incubating at room temperature for 5 min to 60 min.Step two of the method adds 1 ml reagent B to the step one mixture,followed by brief mixing and reading at 485 nm. This negative orindirect assay quantitates protein by the difference in absorbance inthe pre-versus post-bathocuproine chelated sample. It is thus lessaccurate than a positive or direct assay that quantitates proteindirectly. It also uses a large volume of standard protein to reagent(volume standard protein to reagent A is 1:1.6 to 1:2.4).

The method provided herein overcomes such drawbacks and providesadditional benefits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows peptide quantitation using a method according to certainembodiments provided herein compared to the commercial MICRO BCA™method.

FIG. 2 shows a digest quantitation using a method according to certainembodiments provided herein compared to the commercial MICRO BCA™method.

FIG. 3 shows another peptide quantitation using a method according tocertain embodiments provided herein compared to the commercial MICROBCA™ method.

FIG. 4 shows peptide quantitation and variability using an embodiment ofthe methods provided herein.

FIGS. 5A-B show peptide quantitation and variability using otherembodiments of the methods provided herein.

FIGS. 6A-B show effects of varying acetonitrile concentration using oneembodiment of the methods provided herein with a BSA digest and HeLadigest.

FIGS. 7A-B show effect of acetonitrile of measuring peptideconcentration using certain embodiments of the methods provided herein.

FIG. 8 shows peptide quantitation using an embodiment of the methodsprovided herein and a fluorescamine-based assay.

FIGS. 9A-B compares peptide quantitation using an embodiment of themethods provided herein and an indirect two-step method (Matsushita etal., Determination of Proteins by a Reverse Biuret Method Combined withthe Copper-Bathocuproine Chelate Reaction. Clinica Chimica Acta. 216(1993) 103-111).

FIG. 10 demonstrates the ability of one embodiment of the methodsprovided herein to quantitate labeled peptides from a tryptic proteindigest.

FIGS. 11A-B demonstrate quantitation of individual peptides or complexdigests that can be used to aid in evaluation and normalization ofsamples before MS analysis.

SUMMARY

One non-limiting use of the methods provided herein is for MSquantitation of peptides or peptide mixtures. The methods according tothe present disclosure use small sample volumes, and result in a robustsignal with high signal to noise ratio (S/N) compared to other methods.The methods provided herein accurately quantitate complex peptidesamples, such as those generated by tryptic digestion of proteins, celllysates, plasma or serum samples, with low variability.

One embodiment provided herein is a direct method for determiningpeptide or protein concentration in a sample, the method comprising

(a) combining the sample with a quantitation assay reagent compositioncomprising a complex to form a mixture where the complex comprises thefollowing general formula:

where

each of R₁ and R₂ is independently alkyl including but not limited to aC₁-C₆ straight or branched alkyl or a C₆-C₂₀ aryl, alkylaryl, orarylalkyl such as methyl (—CH₃), ethyl (—CH₂CH₃), propyl (—CH₂CH₂CH₃),butyl (—CH₂CH₂CH₂CH₃) or phenyl (—C₆-C₅);

each of R₃ and R₄ is independently selected from the group consisting ofhydrogen (H), sulfonate (—SO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li⁺); phosphonate (—PO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺)or lithium (Li⁺); and carboxylate (—CO₂) salt of sodium (Na⁺), potassium(K⁺) or lithium (Li⁺); and

each of R₃ and R₆ is

where R₇ is independently selected from the group consisting of hydrogen(H), sulfonate (—SO₃) salt of sodium (Na⁺), potassium (K⁺) or lithium(Li⁺); phosphonate (—PO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li⁺); and carboxylate (—CO₂) salt of sodium (Na⁺), potassium(K⁺) or lithium (Li⁺);

with the proviso that at least one of R₃, R₄, and R₇ is not H;

(b) incubating the mixture under conditions sufficient to form a coloredcomplex; and

(c) measuring absorbance of the colored complex at 450 nm to 500 nm as adirect indicator of peptide or protein concentration in the sample.

The method may further comprise, after (c), determining the peptide orprotein concentration in the sample by comparing the directly measuredabsorbance with the absorbance of at least one sample containing a knownconcentration of a standard. The sample may be a biological sample. Thesample may be plasma or serum. The standard can be a peptide, a peptidemixture, or a protein digest. The method may be performed prior to massspectrometry analysis of the sample. The reagent composition in (a) mayfurther comprise tartrate and copper sulfate. The incubation temperaturein (b) may range from room temperature to about 45° C., or from about19° C. to about 22° C., or may be about 37° C., or may be about 45° C.The sample may be a plurality of peptides. The sample volume may beabout 5 μl, may not exceed about 200 μl, may range from about 5 μl toabout 20 μl, from about 10 μl to about 20 μl, or from about 15 μl toabout 20 μl. Absorbance may be determined by an automated microplatereader. The standard may be a peptide, a peptide mixture, or a peptidedigest at a known concentration. The sample may be in a solvent that isan aqueous solvent, an organic solvent, or an aqueous and organicsolvent. The method may further comprise, after (c), analyzing thepeptide(s) by mass spectrometry. An assay component may contain at leastone of an organic solvent or a detergent to improve peptide or proteinsolubility. In one embodiment the reagent composition isbathocuproinedisulfonic acid disodium salt hydrate with about 50%acetonitrile. Absorbance may be read at about 480 nm.

Another embodiment provided herein is a direct method for determiningpeptide or protein concentration in a sample comprising:

(a) combining the sample with a quantitation assay reagent compositioncomprising a complex with 50% acetonitrile to form a mixture, where thecomplex comprises the following general formula:

where

each of R₁ and R₂ is independently alkyl including but not limited to aC₁-C₈ straight or branched alkyl or a C₆-C₂₀ aryl, alkylaryl, orarylalkyl such as methyl (—CH₃), ethyl (—CH₂CH₃), propyl (—CH₂CH₂CH₃),butyl (—CH₂CH₂CH₂CH₃) or phenyl (—C₆H₅);

each of R₃ and R₄ is independently selected from the group consisting ofhydrogen (H), sulfonate (—SO₃) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li⁺); phosphonate (—PO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺)or lithium (Li⁺); and carboxylate (—CO₂) salt of sodium (Na⁺), potassium(K⁺) or lithium (Li⁺); and

-   -   each of R₅ and R₆ is

where R₇ is independently selected from the group consisting of hydrogen(H), sulfonate (—SO₃) salt of sodium (Na⁺), potassium (K⁺) or lithium(Li⁺); phosphonate (—PO₃) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li⁺); and carboxylate (—CO₂) salt of sodium (Na⁺), potassium(K⁺) or lithium (Li⁺);

with the proviso that at least one of R₃, R₄, and R₇ is not H;

(b) incubating the mixture under conditions sufficient to form a coloredcomplex; and

(c) measuring absorbance of the colored complex at 450 nm to 500 nm as adirect indicator of peptide or protein concentration in the sample.

The method may further comprise, after (c), determining the peptide orprotein concentration in the sample by comparing the directly measuredabsorbance with the absorbance of at least one sample containing a knownconcentration of a standard. The standard may be a peptide, a peptidemixture, or a protein digest. The method may be performed prior to massspectrometry analysis of the sample. The reagent composition in (a) mayfurther comprises tartrate and copper sulfate. The incubationtemperature in (b) may range from room temperature to about 45° C. orfrom about 19° C. to about 22° C., or may be about 37° C., or may beabout 45° C. The sample volume may range from about 5 μl to about 20 μl.The sample may be a plurality of peptides. The sample volume may beabout 5 μl, or may range from about 10 μl to about 20 μl, or from about15 μl to about 20 μl, or may not exceed about 200 μl. Absorbance may bedetermined by an automated microplate reader. The standard may be apeptide, a peptide mixture, or a peptide digest at a knownconcentration. In the method, after (c), the peptide(s) may be analyzedby mass spectrometry. In one embodiment, the reagent composition isbathocuproinedisulfonic acid disodium salt hydrate. Absorbance may beread at about 480 nm. The sample may be a biological sample. The samplemay be serum or plasma sample.

Another embodiment provided herein is a peptide quantitation reagentcomposition comprising at least one excipient and

(a) a complex at a concentration ranging from about 0.04 M to about 0.1M where the complex comprises the following general formula:

where

each of R1 and R2 is independently alkyl including but not limited to aC1-C6 straight or branched alkyl or each of R₁ and R₂ is independentlyalkyl including but not limited to a C₁-C₆ straight or branched alkyl ora C₆-C₂₀ aryl, alkylaryl, or arylalkyl such as methyl (—CH₃), ethyl(—CH₂CH₃), propyl (—CH₂CH₂CH₃), butyl (—CH₂CH₂CH₂CH₃) or phenyl(—C₆-C₅);

each of R₃ and Ra is independently selected from the group consisting ofhydrogen (H), sulfonate (—SO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li+); phosphonate (—PO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺)or lithium (Li⁺); and carboxylate (—CO₂ ⁻) salt of sodium (Na⁺),potassium (K⁺) or lithium (Li⁺); and

each of R₅ and R₆ is

where R₇ is independently selected from the group consisting of hydrogen(H), sulfonate (—SO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) or lithium(Li⁺); phosphonate (—PO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li⁺); and carboxylate (—CO₂ ⁻) salt of sodium (Na⁺), potassium(K⁺) or lithium (Li⁺);

with the proviso that at least one of R₃, R₄, and R₇ is not H;

(b) tartrate at a concentration ranging from about 5.7 mM to about 22.7mM, and

(c) copper sulfate at a concentration ranging from about 0.25 mM toabout 0.5 mM, resulting in a peptide quantitation reagent composition.

Another embodiment provided herein is a peptide quantitation reagentcomposition comprising at least one excipient and

(a) a complex at a concentration ranging from about 0.04 M to about 0.1M in 50% acetonitrile, where the complex comprises the following generalformula:

where

each of R₁ and R₂ is independently alkyl including but not limited to aC₁-C₅ straight or branched alkyl or a C₆-C₂₀ aryl, alkylaryl, orarylalkyl such as methyl (—CH₃), ethyl (—CH₂CH₃), propyl (—CH₂CH₂CH₃),butyl (—CH₂CH₂CH₂CH₃) or phenyl (—CH₆H₅);

each of R₃ and R₄ is independently selected from the group consisting ofhydrogen (H), sulfonate (—SO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li⁺); phosphonate (—PO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺)or lithium (Li⁺); and carboxylate (—CO₂ ⁻) salt of sodium (Na⁺),potassium (K⁺) or lithium (Li⁺); and

each of R₅ and R₆ is

independently selected from the group consisting of hydrogen (H),sulfonate (—SO₃) salt of sodium (Na⁺), potassium (K⁺) or lithium (Li⁺);phosphonate (—PO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) or lithium(Li⁺); and carboxylate (—CO₂ ⁻) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li⁺);

with the proviso that at least one of R₃, R₄, and R₆ is not H;

(b) tartrate at a concentration ranging from about 5.7 mM to about 22.7mM, and

(c) copper sulfate at a concentration ranging from about 0.25 mM toabout 0.5 mM, resulting in a peptide quantitation reagent composition.

Another embodiment provided herein is a peptide quantitation reagent kitcomprising instructions for mass spectroscopy quantitation of peptidesusing the kit, and reagents comprising

(a) a complex comprising the following general formula:

where

each of R₁ and R₂ is independently alkyl including but not limited to aC₁-C₆ straight or branched alkyl or a C₆-C₂₀ aryl, alkylaryl, orarylalkyl such as methyl (—CH₃), ethyl (—CH₂CH₃), propyl (—CH₂CH₂CH₃),butyl (—CH₂CH₂CH₂CH₃) or phenyl (—C₆H₅);

each of R₃ and R₄ is independently selected from the group consisting ofhydrogen (H), sulfonate (—SO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li⁺); phosphonate (—PO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺)or lithium (Li⁺); and carboxylate (—CO₂ ⁻) salt of sodium (Na⁺),potassium (K⁺) or lithium (Li⁺); and

each of R₅ and R₆ is

independently selected from the group consisting of hydrogen (H),sulfonate (—SO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) or lithium(Li⁺); phosphonate (—PO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li⁺); and carboxylate (—CO₂ ⁻) salt of sodium (Na⁺), potassium(K⁺) or lithium (Li⁺);

with the proviso that at least one of R₃, R₄, and R₇ is not H;

(b) tartrate, and

(c) copper sulfate,

the composition having pH ranging from about pH 12 to about pH 13.

Another embodiment provided herein is a peptide quantitation reagent kitcomprising instructions for mass spectroscopy quantitation of peptidesusing the kit, and reagents comprising

(a) a complex in 50% acetonitrile, where the complex contains thefollowing general formula:

where

each of R₁ and R₂ is independently alkyl including but not limited to aC₁-C₆ straight or branched alkyl or a C₆-C₂₀ aryl, alkylaryl, orarylalkyl such as methyl (—CH₃), ethyl (—CH₂CH₃), propyl (—CH₂CH₂CH₃),butyl (—CH₂CH₂CH₂CH₃) or phenyl (—C₆H₅);

each of R₃ and R₄ is independently selected from the group consisting ofhydrogen (H), sulfonate (—SO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li⁺); phosphonate (—PO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺)or lithium (Li⁺); and carboxylate (—CO₂ ⁻) salt of sodium (Na⁺),potassium (K⁺) or lithium (Li⁺); and

each of R₅ and R⁶ is

independently selected from the group consisting of hydrogen (H),sulfonate (—SO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) or lithium(Li⁺); phosphonate (—PO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li⁺); and carboxylate (—CO₂ ⁻) salt of sodium (Na⁺), potassium(K⁺) or lithium (Li⁺);with the proviso that at least one of R₃, R₄, and R₇ is not H;

(b) tartrate, and

(c) copper sulfate,

the composition having pH ranging from about pH 12 to about pH 13.

Another embodiment provided herein is a direct method for determiningpeptide or protein concentration in a sample, the method comprising:

(a) combining the sample with a quantitation assay reagent compositioncomprising a complex to form a mixture where the complex is

(b) incubating the mixture under conditions sufficient to form a coloredcomplex; and

(c) measuring absorbance of the colored complex at 450 nm to 500 nm as adirect indicator of peptide or protein concentration in the sample.

The method may further comprise, after (c), determining the peptide orprotein concentration in the sample by comparing the directly measuredabsorbance with the absorbance of at least one sample containing a knownconcentration of a standard. The sample may be a biological sample. Thesample may be plasma or serum. The standard can be a peptide, a peptidemixture, or a protein digest. The method may be performed prior to massspectrometry analysis of the sample. The reagent composition in (a) mayfurther comprises tartrate and copper sulfate. The incubationtemperature in (b) may range from room temperature to about 45° C., orfrom about 19° C. to about 22° C., or may be about 37° C., or may beabout 45° C. The sample may be a plurality of peptides. The samplevolume may be about 5 μl, may not exceed about 200 μl, may range fromabout 5 μl to about 20 μl, from about 10 μl to about 20 μl, or fromabout 15 μl to about 20 μl. Absorbance may be determined by an automatedmicroplate reader. The standard may be a peptide, a peptide mixture, ora peptide digest at a known concentration. The sample may be in asolvent that is an aqueous solvent, an organic solvent, or an aqueousand organic solvent. The method may further comprise, after (c),analyzing the peptide(s) by mass spectrometry. An assay component maycontain at least one of an organic solvent or a detergent to improvepeptide or protein solubility. In one embodiment the reagent compositionis bathocuproinedisulfonic acid disodium salt hydrate with about 50%acetonitrile. Absorbance may be read at about 480 nm.

Another embodiment provided herein is a direct method for determiningpeptide or protein concentration in a sample comprising:

(a) combining the sample with a quantitation assay reagent compositioncomprising a complex with 50% acetonitrile to form a mixture, where thecomplex contains

(b) incubating the mixture under conditions sufficient to form a coloredcomplex; and

(c) measuring absorbance of the colored complex at 450 nm to 500 nm as adirect indicator of peptide or protein concentration in the sample.

The method may further comprise, after (c), determining the peptide orprotein concentration in the sample by comparing the directly measuredabsorbance with the absorbance of at least one sample containing a knownconcentration of a standard. The standard may be a peptide, a peptidemixture, or a protein digest. The method may be performed prior to massspectrometry analysis of the sample. The reagent composition in (a) mayfurther comprises tartrate and copper sulfate. The incubationtemperature in (b) may range from room temperature to about 45° C. orfrom about 19° C. to about 22° C., or may be about 37° C., or may beabout 45° C. The sample volume may range from about 5 μl to about 20 μl.The sample may be a plurality of peptides. The sample volume may beabout 5 μl, or may range from about 10 μl to about 20 μl, or from about15 μl to about 20 μl, or may not exceed about 200 μl. Absorbance may bedetermined by an automated microplate reader. The standard may be apeptide, a peptide mixture, or a peptide digest at a knownconcentration. In the method, after (c), the peptide(s) may be analyzedby mass spectrometry. In one embodiment, the reagent composition isbathocuproinedisulfonic acid disodium salt hydrate. Absorbance may beread at about 480 nm. The sample may be a biological sample. The samplemay be serum or plasma sample.

Another embodiment provided herein is a peptide quantitation reagentcomposition comprising at least one excipient and

(a) a complex at a concentration ranging from about 0.04 M to about 0.1M where the complex contains

(b) tartrate at a concentration ranging from about 5.7 mM to about 22.7mM, and

(c) copper sulfate at a concentration ranging from about 0.25 mM toabout 0.5 mM, resulting in a peptide quantitation reagent composition.

Another embodiment provided herein is a peptide quantitation reagentcomposition comprising at least one excipient and

(a) a complex at a concentration ranging from about 0.04 M to about 0.1M in 50% acetonitrile, where the complex contains

(b) tartrate at a concentration ranging from about 5.7 mM to about 22.7mM, and

(c) copper sulfate at a concentration ranging from about 0.25 mM toabout 0.5 mM, resulting in a peptide quantitation reagent composition.

Another embodiment provided herein is a peptide quantitation reagent kitcomprising instructions for mass spectroscopy quantitation of peptidesusing the kit, and reagents comprising

(a) a complex containing

(b) tartrate, and

(c) copper sulfate,

the composition having pH ranging from about pH 12 to about pH 13.

Another embodiment provided herein is a peptide quantitation reagent kitcomprising instructions for mass spectroscopy quantitation of peptidesusing the kit, and reagents comprising (a) a complex in 50%acetonitrile, where the complex contains

(b) tartrate, and

(c) copper sulfate,

the composition having pH ranging from about pH 12 to about pH 13.

These and other embodiments will be further described as follows.

DETAILED DESCRIPTION

Bathocuproinedisulfonic acid disodium salt hydrate has 500% greatersolubility in water compared to bathocuproine or neocuprione. Theincreased solubility allows higher concentrations of chelator insolution which ultimately provides better sensitivity.

The methods described herein provide increased sensitivity of peptideconcentration determination. In one embodiment, sensitivity was as lowas ok 25 μg/ml. In one embodiment, sensitivity was as low as 15 μg/mlpeptide. In one embodiment, sensitivity ranged from 12.5 μg/ml-1000μg/ml. The methods provided herein do not require peptide denaturationprior to performing the assay. Peptide denaturation on very small samplevolumes is highly variable and time consuming. Eliminating the need fordenaturation results in shorter assay duration and less assayvariability.

The methods according to the present disclosure are positive or directassays. They require a volume of about 200 μl, comprised of a samplevolume from 10 μl to 20 μl, and a reagent volume of 180 μl. The methodsprovided herein allow higher levels of sensitivity to be achieved at asmaller sample volume compared to other commercial assays. The smallervolume enables the methods provided herein to be performed in amulti-well plate and to be read by an automatic reader, versus readingin cuvettes that typically require several, 0.5 ml to 2 mls of sample.The methods described herein permit use of much smaller sample volumesand smaller total volumes compared with the MICRO BCA™ method. The MICROBCA™ assay requires 150 μl sample volume and 300 μl total volume. Thepreviously described “negative” bathocuproine assay requires 50 μlsample volume and 1.15 ml total volume. In favorable contrast, themethods according to the present disclosure require 20 μl maximum samplevolume and 200 μl total volume.

Like other copper based protein assays, certain amino acids in a peptidecan increase or decrease the sensitivity of the methods. Peptidesolubility and structure can also have an influence on the results. Themethods provided herein are best used to quantitate peptides in complexsamples containing a mixture of peptides where differences in peptidelength and sequence are averaged out in the assay. This is of particularimportance in measuring recovery from peptide fractionation methods orstandardizing HPLC load amounts for quantitative proteomic analysis. Themethods disclosed herein can also be used to assay samples containing asingle peptide, however, this can result in increased error, resultingfrom a larger variation in assay response among different peptidescompared to a standard. This can be compensated for by using standardsmore specific to the peptide of interest.

The methods provided herein are one-step (mix and read), and absorbanceis read at 450 nm to 500 nm, in one embodiment at 480 nm.

The methods described herein provide for multiple peptide assays thatallow detection and quantification of complex peptide mixtures beforestandard MS analysis in about 30 minutes.

The methods described herein provide 2-3 fold enhanced signal tobackground ratio (S/B) than the MICRO BCA™ assay at same sample volumes.

The methods provided herein exhibit decreased peptide to peptidevariability due to solubilizing hydrophobic peptides by adding anorganic co-solvent such as acetonitrile, detergents such as SDS andurea, and other water miscible organic solvents.

In certain embodiments, the peptide quantitation methods provided hereinuse a compound as chelator having the following general formula

where

each of R₁ and R₂ is independently alkyl including but not limited to aC₁-C₆ straight or branched alkyl or a C₆-C₂₀ aryl, alkylaryl, orarylalkyl such as methyl (—CH₃), ethyl (—CH₂CH₃), propyl (—CH₂CH₂CH₃),butyl (—CH₂CH₂CH₂CH₃) or phenyl (—C₆H₅);

each of R₃ and R₄ is independently selected from the group consisting ofhydrogen (H), sulfonate (—SO₃) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li⁺); phosphonate (—PO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺)or lithium (Li⁺); and carboxylate (—CO₂ ⁻) salt of sodium (Na⁺),potassium (K⁺) or lithium (Li⁺);

each of R₅ and R₆ is

where R₇ is independently selected from the group consisting of hydrogen(H), sulfonate (—SO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) or lithium(Li⁺); phosphonate (—PO₃ ⁻) salt of sodium (Na⁺), potassium (K⁺) orlithium (Li⁺); and carboxylate (—CO₂ ⁻) salt of sodium (Na⁺), potassium(K⁺) or lithium (Li⁺);

with the proviso that at least one of R₃, R₄, and R₇ is not H.

In one embodiment, the peptide quantitation methods provided herein useas chelator bathocuproinedisulfonic acid disodium salt hydrate shownbelow:

Without being held to a single theory, the complex increases thesolubility of the bathocuproine, resulting in enhanced performance inthe methods provided herein compared to previous methods.

The methods provided herein contain a higher amount of Cu⁺² (80 mg/ml).The methods provided herein reduce Cu⁺² to Cu⁺ as in the BCA method, butin the instant methods the bathocuproine ion then combines with Cu⁺forming a green/orange complex with an absorbance in the range of 450 nmto 500 nm, in one embodiment at 480 nm.

The absorbance-based methods according to certain embodiments providedherein were evaluated with different peptides and protein digests. Assayconditions such as peptide concentration, detection reagentconcentration, pH, incubation time, temperature and the ratio ofdifferent components were optimized. The peptides were assayed on amulti-well plate (e.g., 96 well plate) and the measurements wereconducted using a spectrophotometer to measure absorbance at 480 nm.

EXAMPLE

In one embodiment, 20 μl sample is added to a prepared or pre-madeworking reagent (which is made by mixing 6.9 mM sodium tartratedihydrate, 0.04 M bathocuproinedisulfonic acid disodium salt, and 0.32 Mcopper sulfate). The solution is mixed for one minute, incubated for 30minutes at 37° C. and absorbance is measured at 480 nm.

A working solution is prepared by combining three solutions. Solution 1is prepared by mixing sodium tartrate, sodium carbonate, sodiumhydroxide, and sodium bicarbonate into water. Solution 2 is prepared bymixing bathocuproinedisulfonic acid, disodium salt hydrate into wateroptionally containing acetonitrile. Solution 3 is prepared by mixingcupric sulfate pentahydrate into water. The working solution is formedby combining solutions 1, 2, and 3. Specifically, solution 1 is preparedby mixing, into 50 ml water, 80 mg sodium tartrate (6.9 mM), 3.42 gsodium carbonate (0.65 M), 0.525 ml sodium hydroxide, and 0.89 g sodiumbicarbonate (0.22 M). Solution 2 is prepared by mixing, into 50 ml 50%acetonitrile (25 ml acetonitrile, 25 ml water), 1.13 gbathocuproinedisulfonic acid disodium salt hydrate (0.04 M). Solution 3is prepared by mixing, into 2 ml water, 160 mg cupric sulfatepentahydrate (0.32 M).

The working solution is formed by combining 50 parts Solution 1, 48parts Solution 2, and 2 parts Solution 3.

A volume of 5 μl to 20 μl, typically 20 μl, of standard or samplecontaining peptides or protein is combined with the working solution,shaken for one minute, and incubated from about 22° C. to about 45° C.for, preferably 37° C., for 30 minutes. In one embodiment, 20 μlstandard or sample is combined with 180 μl working solution, shaken forone min, and incubated at 45° C. for 30 minutes. In one embodiment,incubation is at 37° C. After incubation, absorbance at 480 nm ismeasured. The pH range of the final assay mixture is about pH 12 to 13.

While automated absorbance readings, using automated plate readers, aredesirable for speed and convenience, the absorbance measurement of thereaction mixture measured in a cuvette may still be used.

In one embodiment, the methods provided herein quantitate peptides to besubjected to MS analysis. Peptides may include mass tags such as TMT orother covalent modifications used in MS. Peptides to be quantitated mayinclude one or more heavy isotope labeled amino acids.

In one embodiment, the methods provided herein use single peptide ordigested protein standard to generate a standard curve for peptideand/or protein quantitation. In another embodiment, a protein standardis used to generate a standard curve for protein quantitation. Theaccuracy and precision by which a standard curve is generated is crucialfor accurate and precise sample measurement. In one embodiment, astandard curve comprises a mixture of peptides generated from a trypticdigest of bovine serum albumin (BSA) or Protein A/G using methods knownin the art. Choice of a standard that produces an assay response closeto the average for the sample type being analyzed produces the mostaccurate results.

Another embodiment provides for kits containing reagents andinstructions for performing the methods described herein. For example,the kit may contain some or all of the reagents, and instructions,needed to prepare the working solution; one or more standards orinstructions to prepare the standards; instructions to perform theassay; microwell plates, etc.

Over 30 different peptides, peptide mixtures, digests, and samplesconsistent with standard MS analysis were evaluated and are, summarizedin Table 1. A majority (over 70%) of the samples showed reproducible andlinear results.

TABLE 1  SEQ MW Ref. ID Peptide Sequence (Da) # NO w/o LysineVIP (1-12), human, porcine, rat HSDAVFTDNYTR 1425.5 W1 1Beta-Amyloid (1-12) DAEFRHDSGYEV 1424.5 W2 2 Beta-Amyloid (1-8)-CysDAEFRHDSC 1079.1 W3 3 Beta-Amyloid (1-13) DAEFRHDSGYEVH 1561.6 W4 4mid-Lysine Amyloid Precursor Protein (APP) SEVKMDAEFR 1211.4 M1 5(667-676) ACTH (1-17) SYSMEHFRWGKPVGKKR 2093.4 M2 6 Beta-Amyloid (4-17)FRHDSGYEVHHQKL 1752.9 M3 7 Histatin-8 [Hemagglutination- KFHEKHHSHRGY1562.7 M4 8 Inhibiting Peptide (HIP)] C-terminus Lysine VSV-G PeptideYTDIEMNRLGK 1339.5 E1 9 Beta-Amyloid (3-16) EFRHDSGYEVHHQK 1768.9 E2 10Beta-Amyloid (1-16) DAEFRHDSGYEVHHQK 1955 E3 11Antennapedia Peptide, acid RQIKIWFQNRRMKWKK 2246.8 E4 12CEF26, Influenza Virus NP  ILRGSVAHK 980.2 E5 13 (265-274)MOG (35-55), mouse, rat MEVGWYRSPFSRWHLYR 2582 E6 14 NGK w/o LysineACTH (1-10) SYSMEHFRWG 1299.4 W5 15 [Ile-Ser]-Bradykinin (T-Kinin)ISRPPGFSPFR 1260.5 W6 16 Leptin (93-105), human NVIQISNDLENLR 1527.7 W717 mid-Lysine Beta-Amyloid (10-20) YEVHHQKLVFF 1446.7 M5 18[Lys8,9]-Neurotensin (8-13) KKPYIL 761 M6 19 OVA (257-264) SIINFEKL963.2 M7 20 c-terminus Lysine Dynorphin A (2-13), porcine GGFLRRIRPKLK1440.8 E7 21 Beta-Amyloid (18-28) VFFAEDVGSNK 1212.3 E8 22

The methods described herein using bathocuproinedisulfonic acid disodiumsalt hydrate method are sensitive down to 25 μg/ml peptide. Thissensitivity substantially exceeds that of the MICRO BCA™ assay, which ismore sensitive than the standard BCA method. Sensitivity is shown inFIGS. 1-3 quantitating peptide W1 (FIG. 1), BSA digest (FIG. 2) andinsulin (FIG. 3), respectively. Limit of quantitation (LOQ) wascalculated using the equation (10×(st. dev. of blank+avg. of blank)) andthe LOQ for the method described herein and the MICRO BCA™ assay was21.58 μg/ml and 77.40 μg/ml respectively.

Standard solution, peptide samples, or protein digests were loaded at 20μl/well into microplates. Assay working reagent was added at 180 μl/wellworking solution. The plate was sealed, quickly mixed and incubated for30 minutes at 22° C.-45° C. Absorbance was read at 480 nm using aSPECTRAMAX™ Plus384 or VARIOSKAN™ Flash (Type 3001) microplate reader.The established working peptide concentration range for the absorbanceassays was 25 μg/ml -1000 μg/ml. Sample volumes of 5 μl-20 μl were usedallowing for <1 μg peptide to be loaded for an assay sample for theabsorbance assay.

The method described herein demonstrated 3-4 fold increase in S/N,compared to the MICRO BCA™ assay for all peptides tested. The averageconcentration at the limit of quantification (LOQ) was about 15 μg/ml.

The method described herein provides a robust signal and thus results inimproved signal/background (S/B) ratio, which is the ratio of absorbanceat 480 nm at a particular concentration to the absorbance at 480 nm of ablank. This is obtained, e.g., by the following calculation:

${{Signal}\text{/}{Background}\mspace{11mu} \left( {S\text{/}B} \right)\mspace{14mu} {at}\mspace{14mu} 500\mspace{14mu} {µg}\text{/}{mL}} = \frac{A_{480}\mspace{14mu} 0f\mspace{14mu} 500\mspace{14mu} {µg}\text{/}{mL}\mspace{14mu} {sample}}{A_{480}\mspace{14mu} {of}\mspace{14mu} {blank}}$

As shown in FIG. 1 for peptide W1, the present method yielded S/B of311, compared to the MICRO BCA™ assay S/B of 87. Similarly, as shown inFIG. 2 for BSA digest, the present method yielded S/B of 201 compared tothe MICRO BCA™ assay S/B of 55. For insulin (FIG. 3), the present methodyielded S/B of 269 compared to the MICRO BCA™ assay S/B of 81. Thepresent method showed a 3-4 fold increase in signal over the MICRO BCA™assay. These peptides differed in peptide length and hydrophobicitywhich resulted in different sensitivity; Table 1 provides exactsequences.

The variation range in signal intensity among different peptides wasreasonable. Optimizations, such as using an organic solvent and/orhigher incubation temperatures, further decreased variability. Otherorganic solvents and/or detergents such as SDS may also reducevariability. For example, as shown in FIG. 4, a range of variouspeptides were quantitated using 0.04 M bathocuproinedisulfonic aciddisodium salt hydrate in water at 37° C., resulting in 39% variability.As shown in FIG. 5A, when the same peptides were quantitated using 0.04M bathocuproinedisulfonic acid disodium salt hydrate in acetonitrile (anorganic solvent) at 45° C., variability was reduced to 28%. The exampleshown in FIG. 5B shows the same peptides quantitated using 0.04 Mbathocuproinedisulfonic acid disodium salt hydrate in 50% acetonitrileat 37° C. The method described herein provided less variability likelydue to better solubilizing and better formulation of bathocuproine dueto the presence of the disodium salt hydrate and optimized incubationconditions using an organic solvent and increased temperature. As anexample, peptides that are hydrophobic add to variability; acetonitrilereduces variability possibly by reducing secondary and tertiary peptidestructures and/or increasing peptide solubility.

In one embodiment, the amount of acetonitrile in the assay compositionwas optimized. FIGS. 6A-B demonstrate the effect of varying amounts ofacetonitrile in the methods provided herein. Acetonitrile concentrationsranging from 0% to 50% were used to prepare 0.04 Mbathocuproinedisulfonic acid disodium salt hydrate solution. Theformulations were then tested on different concentration of HeLa celldigest (15.6 μg/ml protein to 400 μg/ml protein) and BSA digest, whichwere incubated at 45° C. Both digests were prepared by reducing theappropriate protein with DTT, alkylated with IAA, blocked excess IAAwith DTT, digested with trypsin, then cleaned up using a C18 column. Asshown in FIGS. 6A-B, as the percentage of acetonitrile increased in theformulation, the S/N and assay sensitivity improved. There was 68%improvement between no acetonitrile in the bathocuproinedisulfonic aciddisodium salt hydrate formulation and 50% acetonitrile in thebathocuproinedisulfonic acid disodium salt hydrate formulation.

The effect of acetonitrile in detecting single peptides using the methodprovided herein was evaluated. The results are shown in FIG. 7A forpeptide E4 and in FIG. 7B for peptide El. Two solutions (0.04 Mbathocuproinedisulfonic acid disodium salt hydrate in 50% acetonitrilesolution and 0.04 M bathocuproinedisulfonic acid disodium salt hydratein water) were prepared. Thirteen peptides (data shown for peptides E4and E1) were prepared with concentrations from 15.6 μg/ml to 500 μg/ml.Reagent solution (180 μl) was mixed with 20 μl peptide samples andincubated at 37° C. for 30 min.

As shown in FIGS. 7A and 7B, the presence of acetonitrile in theformulation improved the S/N ratio and improved assay sensitivity. Therewas an average of 45% increase in signal intensity obtained withacetonitrile incorporated in the bathocuproinedisulfonic acid disodiumsalt hydrate formulation.

FIG. 8 shows the peptide concentration of three different proteindigests using protein A/G to generate a standard curve. The results wereextremely reproducible even using a fluorescamine assay, which measurespeptide concentration by labeling the N-termini of peptides (ThermoScientific Nano Drop Protocol. Fluorescamine Protein Assay 2008) and thepresent method using the same standards.

Results from the present method (FIG. 9A), a direct one-step method,were compared with the previously described indirect two-step method(FIG. 9B).

The present method used 0.04 M bathocuproinedisulfonic acid disodiumsalt hydrate in 50% acetonitrile-water mixture; the working solution was0.48 bathocuproinedisulfonic acid disodium salt dehydrate solution: 0.5sodium tartrate solution: 0.02 copper sulfate solution.

The indirect two-step method used ascorbic acid as reducing agent toreduce unreacted Cu⁺² to Cu⁺¹ and then subsequently chelatebathocuproine to Cu⁺¹. The amount of bathocuproine in the indirecttwo-step method was 0.8 mmol/liter, about 500 times lower than theconcentration of bathocuproinedisulfonic acid disodium salt dehydrate inthe present method. The two-step indirect method used a much higherassay total volume of 3 ml, compared to an assay total volume of 200 μlin the present method. A comparison of FIGS. 9A and 9B demonstrates thatthe present method showed more linearity and better sensitivity than theindirect two-step method.

In certain embodiments, the methods provided herein may be used toquantitate labeled proteins and/or peptides. For example, labels used inMS, such as TMT, may be added to the protein or peptide. As shown inFIG. 10, the present method similarly detected and quantitated both TMTlabeled and unlabeled HeLa digests. Because the methods provided hereinare based on the reduction of copper, which is largely dependent on thepeptide amide-backbone, they are not affected by peptide modificationssuch as TMT-labeling of amine groups. This is demonstrated in FIG. 10showing identical responses for a TMT-labeled and unlabeled sample. Thisallows load/injection amounts of TMT-labeled samples to be standardizedbetween MS analysis experiments improving results and consistency. Thepresent method was also comparable with most digestion andsolubilization reagents that are typically used in mass spectrometryanalysis, such as triethylamine, urea, dithiothreitol, acetonitrile(shown in FIGS. 5A-B), formic acid, and trifluoroacetic acid.

One embodiment provides a microplate assay for simple, reliable, andsensitive quantitation of individual peptides or complex digests thatcan be used to aid in evaluation and normalization of samples before MSanalysis. Non-small lung carcinoma cell lines were treated with tendifferent sets of conditions, lysed, reduced, alkylated, and digestedwith trypsin before desalting using C18 columns. The concentration ofeach digest was then determined using the bathocuproine-based peptideassay according to the present teachings. The same results weregenerated using the fluorescamine-based assay (Thermo ScientificNanodrop Protocol. Fluorescamine protein assay. 2008) as demonstrated inFIG. 11A. Each peptide sample concentration was then normalized beforelabeling individually with one of the Thermo Scientific TMT1OPLEX™Isobaric labeling reagents. Labeled samples were combined before peptideidentification and relative quantitation using a Thermo ScientificORBITRAP FUSION™ mass spectrometer. After normalization, non-regulatedpeptides displayed ratios of 1:1:1:1:1:1:1:1:1 despite complex samplehandling and preparation, as shown in FIG. 11B.

The methods described herein provided enhanced sensitivity andflexibility compared to current methods, making them an excellent toolfor determining and monitoring the concentration of peptide samples.They required a relatively small sample volume (10 μl-20 μl), yet stillproviding excellent sensitivity with a working peptide concentrationrange of 25 μg/ml-1000 μg/ml. They are useful tools for monitoring incommon MS applications, e.g., they arepeptide amide-backbone dependentallowing for the quantitation of both labeled and unlabeled peptides.

The embodiments shown and described herein are only specific embodimentsand are not limiting in any way. Therefore, various changes,modifications, or alterations to those embodiments may be made withoutdeparting from the spirit of the invention in the scope of the followingclaims. The references cited are expressly incorporated by referenceherein in their entirety.

1-23. (canceled)
 24. A kit for protein or peptide quantitation of asample comprising: (a) a reagent composition comprising sodiumcarbonate; (b) a reagent composition comprising acetonitrile and acomplex having the formula:

wherein each of R₁ and R₂ is independently chosen from C₁-C₆ straightand branched alkyls, C₆-C₂₀ aryl, alkylaryl, and arylalkyls; each of R₃and R₄ is independently chosen from the group consisting of hydrogen;sulfonate salts chosen from sodium, potassium, and lithium; phosphonatesalts chosen from sodium, potassium, and lithium; and carboxylate saltschosen from sodium, potassium, and lithium; and each of R₅ and R₆ is

wherein R₇ is independently chosen from the group consisting ofhydrogen; sulfonate salts chosen from sodium, potassium, and lithium;phosphonate salts chosen from sodium, potassium, and lithium; andcarboxylate salts chosen from sodium, potassium, and lithium; with theproviso that at least one of R₃, R₄, and R₇ is not H; (c) a reagentcomposition comprising copper sulfate; and (d) instructions for thepeptide quantitation determination, wherein the determination comprisescombining the components (a), (b), and (c) with the sample to form acolored complex, wherein acetonitrile is present in the combination at aconcentration ranging from about 10% to about 50%, and wherein theconcentration of peptide or protein in said sample is directlyproportional to the amount of colored complex that forms once the sampleand components are mixed.
 25. The kit of claim 24, wherein the complexis present in the combination at a concentration ranging from about 0.04M to about 0.1 M.
 26. The kit of claim 24, wherein each of R₁ and R₂ isindependently chosen from methyl, ethyl, propyl, butyl, and phenyl. 27.The kit of claim 24, wherein the kit further comprises (e) an internalstandard chosen from peptides, peptide mixtures, and protein digests.28. The kit of claim 24, wherein the complex has the formula:


29. The kit of claim 28, wherein the acetonitrile is present at aconcentration ranging from about 10% to about 30%.
 30. The kit of claim24, wherein the copper sulfate is present at a concentration rangingfrom about 0.25 M to about 0.5 M.
 31. The kit of claim 24, wherein thereagent composition of (a) further comprises tartrate.
 32. The kit ofclaim 31, wherein the tartrate is present at a concentration rangingfrom about 5.7 mM to about 22.7 mM.
 33. The kit of claim 31, where thetartrate is sodium tartarate.
 34. The kit of claim 24, wherein thereagent composition of (a) further comprises sodium bicarbonate.